Project Summary In Type 1 diabetes (T1D), beta cell antigen-reactive T-cells escape negative selection in the thymus, travel to the periphery, and kill insulin-producing pancreatic beta cells. In healthy individuals, central and peripheral immune tolerance mechanisms prevent this type of autoimmune attack. It is not known which level of tolerance fails in cases of T1D. Introduction of an insulin-reactive T-cell receptor (TCR) derived from a T1D patient to the periphery of humanized mice can initiate diabetes, consistent with the notion that escape of autoreactive T-cells from negative selection promotes disease. On the other hand, beta cell antigen-reactive T-cells have also been isolated from the blood of healthy control (HC) individuals, raising the possibility that defective peripheral tolerance mechanisms may promote T1D. Genetic studies have identified over 40 risk variants for T1D, including genes implicated in both central and peripheral tolerance mechanisms. We hypothesize that there are both hematopoietic stem cell (HSC)-intrinsic and thymus-intrinsic genetic variants in T1D individuals that a) lead to the failure of negative selection of diabetogenic T-cell receptors during T-cell development in the thymus and, additionally or alternatively; b) alter the characteristics of autoreactive T-cells to increase their diabetogenicity. We have established a Personalized Immune (PI) mouse model that allows us to generate an adult human's immune system de novo in immunodeficient NOD/LtSz-scid IL2R gamma null mice (NSG) mice receiving patient HSCs and a partially HLA-matched fetal thymus graft. We have also demonstrated that the patient HSCs can be lentivirally transduced to express a specific TCR and subsequently used to reconstitute NSG mice or human thymus tissue in thymic organ culture assays. With these techniques and with access to HSC and thymus tissue from T1D and HC donors, we will study the thymic selection and subsequent peripheral function of insulin-reactive TCRs expressed on T-cells derived from T1D HSCs and selected in a T1D thymus. In these studies, we aim to determine if there is a genetically-predetermined defect in the central tolerance mechanism of negative selection and/or peripheral immune tolerance mechanisms in T1D.